International Journal of Systematic and Evolutionary Microbiology (2009), 59, 1513–1517 DOI 10.1099/ijs.0.004523-0

Deinococcus gobiensis sp. nov., an extremely radiation-resistant bacterium

Menglong Yuan,1,23 Wei Zhang,13 Shiming Dai,1 Jing Wu,1 Yingdian Wang,2 Tianshen Tao,3 Ming Chen1 and Min Lin1

Correspondence 1Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, Min Lin PR China [email protected] 2College of Science, Beijing Normal University, Beijing 100875, PR China 3China Center for Type Culture Collection, Wuhan University, Wuhan 430072, PR China

A Gram-positive, non-motile, spherical, red-pigmented and facultatively anaerobic bacterium, designated strain I-0T, was isolated from a sand sample of the Gobi desert in Xinjiang Autonomous Region, China. Phylogenetic analysis based on 16S rRNA gene sequences indicated that this isolate represents a novel member of the genus , with low sequence similarities (,94 %) to recognized Deinococcus . The major cellular fatty acids

were C16 : 1v7c and C16 : 0. Its polar lipid profile contained several unidentified glycolipids, phosphoglycolipids, phospholipids, pigments and an aminophospholipid. The peptidoglycan type

was Orn–Gly2 (A3b) and the predominant respiratory quinone was MK-8. The DNA G+C content was 65.4 mol%. DNA–DNA relatedness between strain I-0T and ACCC 10492T was 37 %. The strain was shown to be extremely resistant to gamma radiation (.15 kGy) and UV light (.600 J m”2). On the basis of the phylogenetic, chemotaxonomic and phenotypic data presented, strain I-0T represents a novel species of the genus Deinococcus, for which the name Deinococcus gobiensis sp. nov. is proposed. The type strain is I-0T (5DSM 21396T 5CGMCC 1.7299T).

The genus Deinococcus, which was described by Brooks & likely that radiation resistance evolved as a consequence of Murray (1981), comprises 31 species with validly published chronic exposure to non-radioactive forms of DNA names at the time of writing (http://www.bacterio.cict.fr/d/ damage, such as desiccation (Makarova et al., 2001). deinococcus.html). These species have been isolated from a In the course of the study of stress-resistant from wide range of environments, e.g. desert soil (Rainey et al., arid environments, a novel Deinococcus isolate was 2005; de Groot et al., 2005), aquifers (Suresh et al., 2004), obtained from the upper sand layers of the Gobi desert, the plant rhizosphere (Lai et al., 2006), hot springs Xinjiang, China, where bacteria are exposed to cycles of (Ferreira et al., 1997) and airborne dust (Shashidhar & high and low temperatures and to prolonged desiccation. Bandekar, 2006; Weon et al., 2007). Most members of the In this paper, we report on the taxonomic characterization genus are strictly aerobic, have optimum growth tempera- of this radiation-resistant, red-coloured strain, designated tures in the range 25–35 uC and form red, pink, light-pink I-0T, which was obtained from a mixed sand sample. After or reddish colonies. Their extreme resistance to ionizing exposure of the sample to 10 kGy gamma radiation from a radiation (10 kGy), UV light (600 J m22) and desiccation 60Co source (CAIC), it was enriched in 50 ml TGY (years) is a distinctive characteristic of this genus medium (1.0 % peptone, 0.5 % yeast extract, 0.1 % glucose) (Makarova et al., 2007). This resistance has been attributed at 30 uC with shaking at 200 r.p.m. for up to 5 days, to a highly proficient DNA repair system, and it seems followed by isolation of surviving red-colony-forming bacteria on TGY agar plates (TGY medium with 1.5 % 3These authors contributed equally to this work. agar). The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene Morphology of cells grown for 24–48 h on TGY agar was sequence of strain I-0T is EU427464. examined by zoom stereo microscopy (model SZX7; T Micrographs of colonies and cells of strain I-0 , a 16S rRNA gene Olympus), light microscopy (model BX-51; Olympus), sequence-based maximum-parsimony tree, the fatty acid profile of strain scanning electron microscopy (model S-570; Hitachi) and I-0T and a table of 16S rRNA gene sequence similarities to related strains are available as supplementary material with the online version of transmission electron microscopy (model H-7500; this paper. Hitachi). Gram staining was carried out using the modified

004523 G 2009 IUMS Printed in Great Britain 1513 M. Yuan and others method of Cowan (1974). Physiological characterization production and the Voges–Proskauer test. Strain I-0T and additional biochemical tests were performed as could degrade gelatin, starch and casein and utilized a described by Anderson et al. (1956). number of substrates as sole carbon sources for growth. These sole carbon source tests showed distinct results for Whole-cell hydrolysates were prepared as described by strain I-0T and other Deinococcus species (Table 1 and the Hasegawa et al. (1983). Fatty acid methyl esters were species description). analysed by using the trimethyl sulfonium hydroxide method of Butte (1983). Polar lipid analysis, cell-wall The whole-cell sugars contained mainly glucose and small peptidoglycan analysis and quinone analysis were carried quantities of ribose. The major cellular fatty acids were out according to the methods outlined by Zhang et al. straight-chain C16 : 1v7c (42.07 %) and C16 : 0 (35.06 %) (2007) and performed at the Institute of Microbiology of (Table 1, Supplementary Table S1). This combination Yunnan Province. allows strain I-0T to be distinguished from recognized species of the genus Deinococcus. Cell-wall peptidoglycan Genomic DNA of strain I-0T was extracted using a analysis showed that the cell wall contained L-ornithine as TIANamp bacteria DNA kit (Tiangen) according to the the diamino acid (A3b). The major respiratory quinone in manufacturer’s instructions. The 16S rRNA gene was strain I-0T was MK-8, as in all recognized Deinococcus amplified by PCR with bacterial universal primers F27 species. and R1492, which were adapted from primers fD1 and rP1 (Weisburg et al., 1991), and then ligated into the pSURE-T As shown in Fig. 1, the polar lipid profile of strain I-0T vector (Galen), followed by sequencing by Takala Co. consisted of three unidentified glycolipids, four phospho- (Dalian, China). The DNA G+C content was determined glycolipids, three phospholipids, two pigment spots and an by the thermal denaturation method (Marmur & Doty, aminophospholipid. The polar lipid profile of strain I-0T 1962) and was calculated by using the equation of Owen & was dominated by phosphoglycolipids, which co-migrated Lapage (1976). Escherichia coli K-12 CGMCC 1.3065 (DNA with those found in other Deinococcus species (Embley G+C content 50.6 mol%) was used as a control. DNA– et al., 1987; Ferreira et al., 1997; Suresh et al., 2004; Lai DNA hybridization studies were carried out by using the et al., 2006; Rainey et al., 2007; Zhang et al., 2007). The fluorometric microdilution plate method (Ezaki et al., chromatographic behaviour of the polar lipids PL, APL, 1988; Sawabe et al., 1998). PGL1–2 and GL3 and pigments PIG1–2 of strain I-0T was similar to that of the lipid spots PL, APL, PGL1–2 and GL3 To determine resistance of the culture to gamma radiation, and PIG1–2 reported for D. radiodurans AS 1.633T (Zhang cells were harvested by centrifugation at 5000 g for 5 min et al., 2007). The presence of these lipids extracted from and then washed twice with 100 mM potassium phosphate strain I-0T confirms that it should be assigned to the genus buffer (pH 7.0). Four millilitres of the suspension was Deinococcus. exposed to different doses (0, 5, 10 and 15 kGy) of gamma radiation from a 60Co source (6 kGy h21; BNU) in a 15 ml An almost-complete 16S rRNA gene sequence (1470 bp) T EP tube. Irradiated cultures were serially diluted and 100 ml was determined for strain I-0 .AFASTA search of the aliquots were spread on TGY agar plates. After incubation EMBL nucleotide sequence database using this sequence at 30 uC for 3–4 days, the total c.f.u. ml21 was determined. showed relatively low similarity (,94 %) to sequences To determine the resistance of the isolate to UV light, UV from other Deinococcus species (Supplementary Table survival curves were obtained according to the method of S2), which indicated that this strain might represent a Liu et al. (2008). For both experiments, E. coli K-12 novel species. Phylogenetic analyses were performed CGMCC 1.3065 served as a negative control and using MEGA version 4 (Tamura et al., 2007). Deinococcus radiodurans ACCC 10492T served as a positive Phylogenetic dendrograms, which showed slightly differ- control. ent phylogenetic topologies, were conducted by the neighbour-joining (Fig. 2) and maximum-parsimony Strain I-0T was a Gram-positive, non-spore-forming (Supplementary Fig. S4) methods with bootstrap values coccus. It grew well on TGY agar in an incubator and based on 1000 replications. The G+Ccontentofthe could grow on nutrient agar in an anaerobic chamber, but DNA was 65.4 mol%. DNA–DNA hybridization tests did not grow on Luria–Bertani agar. Colonies on TGY indicated that the relatedness between strain I-0T and D. plates were pink–red, circular, opaque and convex with radiodurans ACCC 10492T was 37 %. regular edges (Supplementary Fig. S1, available in IJSEM Online). Cells were 1.0–2.2 mm in diameter, occurring Survival rates after exposure to various doses of gamma singly and in tetrad forms and usually in pairs, and did not radiation and UV light were analysed for strain I-0T, D. form spores (Supplementary Fig. S2). The ultrastructure of radiodurans ACCC 10492T and E. coli K-12 CGMCC strain I-0T was similar to that of ALT- 1.3065 (Fig. 3). The gamma radiation and UV light survival 1bT (Ferreira et al., 1997), which showed a cell wall with curves of E. coli K-12 CGMCC 1.3065 dropped most several layers and a large electron-dense granule sharply, while the two Deinococcus strains were significantly (Supplementary Fig. S3). Strain I-0T was positive for resistant to gamma radiation and UV light. Compared with catalase, oxidase and urease and reduction of nitrate to D. radiodurans ACCC 10492T, strain I-0T showed higher nitrite, but negative for arginine dihydrolase, indole resistance to gamma radiation and UV light.

1514 International Journal of Systematic and Evolutionary Microbiology 59 Deinococcus gobiensis sp. nov.

Table 1. Properties of strain I-0T useful for differentiation from type strains of related species of genus Deinococcus

Strains: 1, strain I-0T (data from this study); 2, D. radiodurans R1T [data for carbon utilization, cell morphology, pigmentation and Gram reaction from this study and data for fatty acids from Zhang et al. (2007) using strain ACCC 10492T; other data from Brooks & Murray (1981)];3,D. hohokamensis KR-40T;4,D. navajonensis KR-114T (data in columns 3 and 4 from Rainey et al., 2005); 5, D. indicus Wt/1aT (Suresh et al., 2004); 6, T T D. deserti VCD115 (de Groot et al., 2005); 7, D. grandis DSM 1963 (Oyaizu et al., 1987; Suresh et al., 2004). +, Positive; 2, negative; W, weakly positive; ND, no data available.

Characteristic 1 2 3 4 5 6 7

Pigmentation Pink–red Pink–red Light pink Pink Red Faintly pink Red or pink Cell morphology Coccus Coccus Coccus Rod Rod Rod Rod Gram reaction ++++222 Demand for oxygen Facultative Strict aerobe Aerobe Aerobe Strict aerobe Strict aerobe Aerobe anaerobe Cytochrome oxidase ++++2 ND 2 Catalase ++222 ++ Carbon source utilization D-Fucose 2 + 2 + ND ND ND Raffinose + W 22+ ND 2 D-Xylose 2222ND 2 ND Fructose ++2 + 2 + ND Glucose ++222 + 2 Lactose + 222+ 22 L-Arabinose 22+++ 22 L-Tryptophan 2 + 22+ 22 Major fatty acids (%) 16 : 1v7c (42.1), 16 : 1v7c (39.9), 16 : 1v7c (ND), 16 : 1v7c (ND), 15 : 1 (12.5), 16 : 1v7c (29.4), 15 : 0 (13), 16 : 0 (35.1) 16 : 0 (14.1), 17 : 1v8c (ND), 17 : 1v8c (ND), 16 : 1 (33.7), 17 : 1v8c (14.1) 15 : 1 (23), 17 : 1v8c (11.8) 17 : 1v7c iso 17 : 1v7c iso 17 : 1 (10.7) 16 : 1 (18) (ND) (ND) DNA G+C content 65.4 67 67.9 66.4 65.8 60 68.7 (mol%)

In summary, the results of 16S rRNA gene sequence comparison and the chemotaxonomic data clearly dem- onstrate that strain I-0T is a member of the genus Deinococcus. On the basis of its distinct phylogenetic position, the presence of the combination of fatty acids C16 : 1v7c and C16 : 0 and its phenotypic characteristics (Table 1), strain I-0T represents a novel species of the genus Deinococcus, for which the name Deinococcus gobiensis sp. nov. is proposed.

Description of Deinococcus gobiensis sp. nov. Deinococcus gobiensis (go.bi.en9sis. N.L. masc. adj. gobiensis pertaining to the Gobi, a great bare-rock desert in Xinjiang Autonomous Region, China, the source of the type strain). Cells are facultatively anaerobic, Gram-positive, non- spore-forming cocci, 1.0–2.2 mm in diameter. Catalase- and oxidase-positive. Reduces nitrate to nitrite. Positive for urease and negative for arginine dihydrolase and indole production. Negative in the Voges–Proskauer test. Grows Fig. 1. Polar lipid profile of strain I-0T. GL3–GL5, Unidentified well on TGY agar at 15–35 uC (optimum 30 uC) and pH 7– glycolipids; PGL1–PGL4, unidentified phosphoglycolipids; PL, 8; does not grow on LB agar. The reddish colonies are PL1 and PL2, unidentified phospholipids; APL, unidentified circular, opaque and convex with regular edges. Glucose, aminophospholipid; PIG1 and PIG2, pigments. sucrose, lactose, fructose, L-aspartic acid and L-histidine http://ijs.sgmjournals.org 1515 M. Yuan and others

Fig. 2. Neighbour-joining phylogenetic tree constructed from a comparative analysis of 16S rRNA gene sequences showing the relationships of strain I-0T with other Deinococcus strains. Numbers on branch nodes are percentage bootstrap values. Bar, 0.02 substitutions per nucleotide position. The sequence of YT-1T was used as an outgroup.

can be utilized as sole carbon sources, but L-arabinose, D- fucose, D-xylose, L-rhamnose, L-tryptophan and L-arginine can not. Gelatin, starch and casein are degraded. The major cellular fatty acids are C16 : 1v7c and C16 : 0. Peptidoglycan type is Orn–Gly2 (A3b). The major respiratory quinone is MK-8. The polar lipid profile consists of various uniden- tified glycolipids, phosphoglycolipids, phospholipids, pig- ments and an aminophospholipid. The DNA G+C content of the type strain is 65.4 mol%. The type strain is extremely resistant to gamma radiation (.15 kGy) and UV light (.600 J m22) compared with E. coli K-12 CGMCC 1.3065 and D. radiodurans ACCC 10492T. The type strain is I-0T (5DSM 21396T 5CGMCC 1.7299T), isolated from a mixed sand sample from the Gobi desert.

Acknowledgements The authors wish to thank Professor Xiuzhu Dong (Institute of Microbiology, Chinese Academy of Sciences) for reading the typescript. This work was supported by the Ministry of Science and Technology of China (National Basic Research Program 2007CB707805 and National High-Tech Program 2007AA021305, 2006AA02Z229 and 2006AA020101) and the National Natural Science Foundation of China (grant no. 30670050).

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